When traversing microvascular beds, such as in the lung, red blood cells (RBCs) are subjected to mechanical deformation. Our previous findings that RBCs are required for flow-induced nitric oxide (NO) synthesis in the lung and that mechanical deformation of RBCs results in the release of adenosine triphosphate (ATP), a stimulus for endothelial NO synthesis, suggested a novel mechanism for the control of vascular resistance in the pulmonary circulation. In this construct, as the RBC is increasingly deformed by increments in the velocity of blood flow through a vessel and/or by reductions in vascular caliber, it releases ATP which stimulates endothelial NO synthesis. The abluminal release of NO results in relaxation of vascular smooth muscle and, consequently, an increase in vascular caliber. This vasodilation results in a decrease in vascular resistance, and, thereby, a decrease in the stimulus for RBC deformation and ATP release. Thus, in response to RBC-derived ATP, abluminal release of NO would indirectly inhibit deformation-induced ATP release from RBCs via effects on vascular caliber. However, NO is also released into the vascular lumen by the endothelium in response to ATP. Therefore, in addition to effects on vascular caliber, it is possible that NO released into the vascular lumen could interact directly with circulating RBCs to modulate deformation-induced ATP release. Here, we address the hypothesis that: Deformation-induced ATP release from RBCs of rabbits and humans is regulated by nitric oxide via effects on the activity of a heterotrimeric G protein. In this proposal we intend to 1) establish that the effects of NO donors on deformation-induced ATP release from these RBCs are related to the release of NO, itself, 2) demonstrate that the ability of nitric oxide to inhibit ATP release from these RBCs is independent of an effect on RBC deformability per se, 3) demonstrate that nitric oxide inhibits deformation-induced ATP release from these RBCs as the result of inactivation of a heterotrimeric G protein of the Gi subclass and 4) demonstrate that the ability of nitric oxide to inhibit deformation-induced ATP release from RBCs of rabbits and humans is the result of the stimulation of mono-ADP-ribosylation (inactivation) of Gi and that this effect is reversible. The successful completion of the studies described in this proposal will define a new role for circulating RBCs as regulators of vascular resistance. This proposal is the logical extension of our previous work and is consistent with a major focus of our group, namely, characterization of those mechanisms responsible for the control of vascular resistance, and specifically, the role of RBC-derived ATP as a stimulus for endogenous NO synthesis.